Biomedical subjects

Thomas Voets

Publications and source records attributed to Thomas Voets.

28 records · Page 2Linked to original sources

PubMed2004

TRPV4 calcium entry channel: a paradigm for gating diversity.

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4alpha-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca2+ entry channels.

Amino Acid Sequence↗

PubMed2004

Diversity of TRP channel activation.

Calcium entry controls a plethora of short- and long-term cell functions. With respect to the activation mechanisms of Ca2+ entry channels they are subdivided in the thoroughly characterized voltage-gated Ca2+ channels and the non-voltage gated Ca2+ channels. The latter group includes cation channels of the 'transient receptor potential' (TRP) superfamily, which consists of three subfamilies (TRPC, TRPV, TRPM). Activation of TRP channels is not yet completely understood, but examples of activation mechanisms of TRP channels from all three subfamilies will be discussed. The main focus is on the members of the TRPV subfamily, among which the TRPV4 channel shows a surprising gating promiscuity. It can be activated by cell swelling, heat and phorbol esters. Endogenous activators of the channel have not yet been described. It will be shown that lipid messengers related to arachidonic acid are endogenous TRPV4 activators. For TRPV5 and 6, the only highly Ca2+ selective channels within the TRP super-family, a voltage-dependent gating mechanism will be discussed, which includes an open pore block by Mg2+ and a highly Ca2+-sensitive mechanism of inactivation. Regulation of channel availability by interaction with a protein bound to a site at the C-terminus of both channels will be demonstrated. Functional consequences of these different mechanisms of gating will be discussed.

Animals↗

PubMed2003

TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption.

Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia.

Amino Acid Sequence↗

PubMed2003

Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels.

TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.

8,11,14-Eicosatrienoic Acid↗

PubMed2003

Voltage dependence of the Ca2+-activated cation channel TRPM4.

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.

Action Potentials↗

PubMed2003

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6.

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.

Aspartic Acid↗

PubMed2002

Molecular determinants of permeation through the cation channel TRPV4.

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

Amino Acid Sequence↗

PubMed2002

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis.

Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.

Amino Acid Substitution↗

PubMed2002

Additional fluxes of activator Ca2+ accompanying Ca2+ release from the sarcoplasmic reticulum triggered by insP3-mobilizing agonists.

Activation of phospholipase C (PLC)-linked receptors leads not only to Ca2+ release from the sarcoplasmic reticulum (SR) by inositol-1,4,5-trisphosphate (InsP3), but also to Ca2+ entry via opening of receptor-activated Ca2+ channels (RACCs) and store-operated Ca2+ channels (SOCs), in addition to possible contributions of Ca2+ release from non-SR stores. We review recent results on these non-SR Ca2+ fluxes. In A7r5 smooth-muscle cells (SMCs), high InSP3 concentrations release Ca2+ from a thapsigargin-insensitive store. Presumably this store corresponds to the Golgi and is filled by a Pmrl-type Ca2+ pump. Molecular candidates for RACCs and SOCs are found among the members of the TRPC channel family. Inoue and colleagues have recently demonstrated that in vascular SMCs TRPC6 is an essential part of a RACC that is activated by alpha-adrenergic stimulation via the diacylglycerol branch of phosphatidylinositol-4,5-bisphosphate hydrolysis. In TRPC4 knockout mice, contractility of SMCs appears unaffected. However, endothelium-dependent relaxation is impaired mainly due to lack of a SOC activity in endothelial cells. The best-characterized SOC current, mainly observed in blood cells, is Icrac Recently, it has been proposed that CaTI (TRPV5) forms at least part of the pore of CRAC. This view is challenged by data from our laboratory.

Animals↗

PubMedDate not supplied

The pore of TRP channels: trivial or neglected?

Ultimate proof that a protein contributes to the pore of an ion channel is demonstrating that pore properties can be altered by mutations to the putative pore-forming region. To date this has only been achieved for a few TRP proteins, and only within the TRPV subfamily. The location and structure of the pore region and selectivity filter of most TRP proteins, including all members of the TRPM and TRPC subfamilies, is currently unknown. In this review we give a short overview of the limited current knowledge about TRP channel pores and argue that further study is needed, not only for a better understanding of the mechanisms underlying cation permeation, but also to establish that all members of the TRP superfamily indeed function as bona fide ion channels.

Amino Acid Sequence↗